Spinal cord injury (SCI) is a devastating event affecting approximately 11,000 individuals each year in the US alone. Although most victims survive, the individual is often paralyzed for the rest of his or her life. Sixty percent of injuries occur in individuals between the ages of 16 and 30. Life-time medical costs average about $1.5 million dollars per patient and combined with lost earning potential make SCI a major burden both on an individual and national level. SCI results in permanent injury in part because neurons fail to regenerate through the glial scar that is formed after injury. This glial scar contains extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABCI [cABCI) is a bacterial enzyme that catalyzes the degradation of chondroitin sulfate carbohydrate chains such as those found on SPGs. Exciting recent work in animal models of SCI has shown that treatment with cABCI dramatically improves both motor and autonomic functional outcomes after injury. Although the benefits observed in animal models are significant and will likely translate to the human condition, chondroitinase therapies are challenged by the relative thermal instability of cABCI at body temperatures. The short enzyme halflife necessitates frequent dosing, limiting the size of animal trials and making human therapy challenging as well. A cABCI enzyme with increased stability at body temperatures will greatly facilitate research and make human therapies safer, easier and more cost effective by allowing use of indwelling pumps and sustained release formulations rather than repeated intrathecal injections. Phase 1 of this grant is designed to improve the thermal stability of cABCI to make it a more useful agent and potential human therapeutic. Given success during this initial work, phase 2 will advance the lead candidate into animal efficacy and preclinical development. Thermal stability of cABCI will be improved through VerdiZyme's Directed Gene Assembly (DGA) technology. This directed evolution (DE) approach has been used previously to increase the thermal stability of numerous enzymes. In this proposal, techniques drawn from several of these successful DE experiments will be used to construct variants of the cABCI enzyme with increased thermal stability through (i) knawledge-based-rational protein redesign, (ft) random mutagenesis, and (Hi) separation of advantageous and detrimental mutations through-recombination. Variants will be screened for enhanced thermal stability and reassorted for optimization with DGA. Lead candidates will be purified-and characterized. The aims of this proposal are 1) Produce a library of up to 100,000 chondroitinase mutants 2) Screen library for enhanced chondroitinase .thermal stability, 3) Re-assort identified aEeles to isolate advantageous combinations and 4) Purify and characterize chondroitinase product candidates. [unreadable] [unreadable] [unreadable]